A need exists for recombinant or modified therapeutic agents having glucagon antagonist activity.
Recombinant and modified proteins are an emerging class of therapeutic agents. Useful modifications of protein therapeutic agents include combination with the xe2x80x9cFcxe2x80x9d domain of an antibody and linkage to polymers such as polyethylene glycol (PEG) and dextran. Such modifications are discussed in detail in a patent application entitled, xe2x80x9cModified Peptides as Therapeutic Agents,xe2x80x9d U.S. Ser. No. 09/428,082, PCT appl. No. WO 99/25044, which is hereby incorporated by reference in its entirety.
A much different approach to development of therapeutic agents is peptide library screening. The interaction of a protein ligand with its receptor often takes place at a relatively large interface. However, as demonstrated for human growth hormone and its receptor, only a few key residues at the interface contribute to most of the binding energy. Clackson et al. (1995), Science 267: 383-6. The bulk of the protein ligand merely displays the binding epitopes in the right topology or serves functions unrelated to binding. Thus, molecules of only xe2x80x9cpeptidexe2x80x9d length (2 to 40 amino acids) can bind to the receptor protein of a given large protein ligand. Such peptides may mimic the bioactivity of the large protein ligand (xe2x80x9cpeptide agonistsxe2x80x9d) or, through competitive binding, inhibit the bioactivity of the large protein ligand (xe2x80x9cpeptide antagonistsxe2x80x9d).
Phage display peptide libraries have emerged as a powerful method in identifying such peptide agonists and antagonists. See, for example, Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science 249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No. 5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar. 12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No. 5,338,665, issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul. 13, 1999; WO 96/40987, published Dec. 19, 1996; and WO 98/15833, published Apr. 16, 1998 (each of which is incorporated by reference in its entirety). In such libraries, random peptide sequences are displayed by fusion with coat proteins of filamentous phage. Typically, the displayed peptides are affinity-eluted against an antibody-immobilized extracellular domain of a receptor. The retained phages may be enriched by successive rounds of affinity purification and repropagation. The best binding peptides may be sequenced to identify key residues within one or more structurally related families of peptides. See, e.g., Cwirla et al. (1997), Science 276: 1696-9, in which two distinct families were identified. The peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401-24.
Another biological approach to screening soluble peptide mixtures uses yeast for expression and secretion (Smith et al. (1993), Mol. Pharmacol. 43: 741-8) to search for peptides with favorable therapeutic properties. Hereinafter, this and related methods are referred to as xe2x80x9cyeast-based screening.xe2x80x9d
Still other methods compete with phage display in peptide research. A peptide library can be fused to the carboxyl terminus of the lac repressor and expressed in E. coli. Another E. coli-based method allows display on the cell""s outer membrane by fusion with a peptidoglycan-associated lipoprotein (PAL). Hereinafter, these and related methods are collectively referred to as xe2x80x9cE. coli display.xe2x80x9d In another method, translation of random RNA is halted prior to ribosome release, resulting in a library of polypeptides with their associated RNA still attached. Hereinafter, this and related methods are collectively referred to as xe2x80x9cribosome display.xe2x80x9d Other methods employ peptides linked to RNA; for example, PROfusion technology, Phylos, Inc. See, for example, Roberts and Szostak (1997), Proc. Natl. Acad. Sci. USA, 94: 12297-303. Hereinafter, this and related methods are collectively referred to as xe2x80x9cRNA-peptide screening.xe2x80x9d Chemically derived peptide libraries have been developed in which peptides are immobilized on stable, non-biological materials, such as polyethylene rods or solvent-permeable resins. Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass slides. Hereinafter, these and related methods are collectively referred to as xe2x80x9cchemical-peptide screening.xe2x80x9d Chemical-peptide screening may be advantageous in that it allows use of D-amino acids and other unnatural analogues, as well as non-peptide elements. Both biological and chemical methods are reviewed in Wells and Lowman (1992), Curr. Opin. Biotechnol. 3: 355-62.
In the case of known bioactive peptides, rational design of peptide ligands with favorable therapeutic properties can be completed. In such an approach, one makes stepwise changes to a peptide sequence and determines the effect of the substitution upon bioactivity or a predictive biophysical property of the peptide (e.g., solution structure). Hereinafter, these techniques are collectively referred to as xe2x80x9crational design.xe2x80x9d In one such technique, one makes a series of peptides in which one replaces a single residue at a time with alanine. This technique is commonly referred to as an xe2x80x9calanine walkxe2x80x9d or an xe2x80x9calanine scan.xe2x80x9d When two residues (contiguous or spaced apart) are replaced, it is referred to as a xe2x80x9cdouble alanine walk.xe2x80x9d The resultant amino acid substitutions can be used alone or in combination to result in a new peptide entity with favorable therapeutic properties.
Structural analysis of proteinxe2x80x94protein interaction may also be used to suggest peptides that mimic the binding activity of large protein ligands. In such an analysis, the crystal structure may suggest the identity and relative orientation of critical residues of the large protein ligand, from which a peptide may be designed. See, e.g., Takasaki et al. (1997), Nature Biotech. 15: 1266-70. Hereinafter, these and related methods are referred to as xe2x80x9cprotein structural analysis.xe2x80x9d These analytical methods may also be used to investigate the interaction between a receptor protein and peptides selected by phage display, which may suggest further modification of the peptides to increase binding affinity.
Conceptually, one may discover peptide mimetics or antagonists of any protein using phage display, RNA-peptide screening, yeast-based screening, rational design, and the other methods mentioned above.
The present invention concerns therapeutic agents that have glucagon antagonist activity with advantageous pharmaceutical characteristics (e.g., half-life). In accordance with the present invention, such compounds comprise:
a) a glucagon antagonist domain, preferably having very little or no glucagon agonist activity, or sequences derived therefrom by rational design, yeast-based screening phage display, RNA-peptide screening, or the other techniques mentioned above; and
b) a vehicle, such as a polymer (e.g., PEG or dextran) or an Fc domain, which is preferred;
wherein the vehicle is covalently attached to the glucagon antagonist domain. The vehicle and the glucagon antagonist domain may be linked through the N- or C-terminus of the glucagon antagonist domain, as described further below. The preferred vehicle is an Fc domain, and the preferred Fc domain is an IgG Fc domain. Preferred glucagon antagonist domains comprise the amino acid sequences described hereinafter in Table 1. Glucagon antagonist domains can be generated by rational design, yeast secretion screening, rational design, protein structural analysis, phage display, RNA-peptide screening and the other techniques mentioned herein.
Further in accordance with the present invention is a process for making therapeutic agents having glucagon antagonist activity, which comprises:
a. selecting at least one peptide having glucagon antagonist activity; and
b. covalently linking said peptide to a vehicle.
The preferred vehicle is an Fc domain. Step (a) is preferably carried out by selection from the peptide sequences in Table 1 hereinafter or from phage display rational design, yeast secretion screening, rational design, protein structural analysis, RNA-peptide screening, or the other techniques mentioned herein.
The compounds of this invention may be prepared by standard synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins. Although non-natural amino acids cannot be expressed by standard recombinant DNA techniques, techniques for their preparation are known in the art. Compounds of this invention that encompass non-peptide portions may be synthesized by standard organic chemistry reactions, in addition to standard peptide chemistry reactions when applicable.
The primary use contemplated for the compounds of this invention is as therapeutic or prophylactic agents. The vehicle-linked peptide may have activity that is able to out compete the natural ligand at reasonable therapeutic doses.
The compounds of this invention may be used for therapeutic or prophylactic purposes by formulating them with appropriate pharmaceutical carrier materials and administering an effective amount to a patient, such as a human (or other mammal) in need thereof. Other related aspects are also included in the instant invention.
Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention.